LOCUS       sequence-230319-       11590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cas9 and sgRNA targeting SHMT2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11590)
  AUTHORS   Minton DR, Nam M, McLaughlin DJ, Shin J, Bayraktar EC, Alvarez SW, 
            Sviderskiy VO, Papagiannakopoulos T, Sabatini DM, Birsoy K, 
            Possemato R
  TITLE     Serine Catabolism by SHMT2 Is Required for Proper Mitochondrial 
            Translation Initiation and Maintenance of Formylmethionyl-tRNAs.
  JOURNAL   Mol Cell. 2018 Feb 15;69(4):610-621.e5. doi: 
            10.1016/j.molcel.2018.01.024.
  PUBMED    29452640
REFERENCE   2  (bases 1 to 11590)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2018.01"; journalName: "Mol Cell"; date: 
            "2018-02-15- 15"; volume: "69"; issue: "4"; pages: "610-621.e5"
COMMENT     Sequence Label: pLenti CRISPR sgSHMT2_2
FEATURES             Location/Qualifiers
     source          1..11590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    105..126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(141..146,147..164,165..171)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    179..195
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     184..206
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     203..219
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     203..219
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     238..258
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        240..258
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        286..512
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             513..693
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    740..865
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1358..1591
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1776..1820
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        1998..2246
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1998..2018
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2169..2188
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2268..2343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2391..2508
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2577..2788
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2808..2817
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2817..2840
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2847..2867
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2892..6992
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2892..6992
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6993..7040
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7056..7112
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7113..7712
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7113..7132)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7609..7629
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7728..8316
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7781..7801)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8199..8210)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8388..8621
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8699..8820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8736..8755)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8790..8809
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8860..8995
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9015..9034)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9016..9034)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9044..9061)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9044..9060)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9053..9075)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9202..9657
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9289..9308)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9499..9520
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9683..9787
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9788..9856,9857..10648)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(10006..10025)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10819..11407
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11308..11327
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11561..11578
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
       61 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      121 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      181 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      241 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
      301 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
      361 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
      421 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
      481 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
      541 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      601 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      661 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      721 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      781 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      841 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      901 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      961 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
     1021 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1081 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1141 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1201 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1261 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     1321 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     1381 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     1441 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     1501 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     1561 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     1621 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     1681 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     1741 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     1801 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     1861 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     1921 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     1981 ccccgagggg acccagagag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2041 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
     2101 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2161 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2221 atatcttgtg gaaaggacga aacaccggta gacggccagg ttggctggtt ttagagctag
     2281 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     2341 tgcttttttg aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga
     2401 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     2461 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     2521 agggacagca gagatccact ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc
     2581 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
     2641 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     2701 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2761 tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc accatggatt
     2821 acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag
     2881 tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct
     2941 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
     3001 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa
     3061 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
     3121 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
     3181 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
     3241 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
     3301 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
     3361 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     3421 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     3481 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     3541 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     3601 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     3661 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     3721 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     3781 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     3841 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     3901 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     3961 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     4021 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     4081 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     4141 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     4201 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     4261 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     4321 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact
     4381 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     4441 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     4501 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
     4561 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     4621 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     4681 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     4741 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     4801 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     4861 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     4921 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     4981 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     5041 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     5101 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     5161 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     5221 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     5281 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     5341 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     5401 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
     5461 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     5521 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     5581 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     5641 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     5701 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     5761 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     5821 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     5881 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     5941 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     6001 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     6061 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     6121 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     6181 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     6241 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     6301 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     6361 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     6421 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     6481 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     6541 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     6601 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     6661 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     6721 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     6781 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     6841 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     6901 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     6961 cacggatcga cctgtctcag ctgggaggcg acaagcgtcc tgctgctact aagaaagctg
     7021 gtcaagctaa gaaaaagaaa gctagcggca gcggcgccac caacttcagc ctgctgaagc
     7081 aggccggcga cgtggaggag aaccccggcc ccatgaccga gtacaagccc acggtgcgcc
     7141 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     7201 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     7261 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     7321 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     7381 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     7441 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag
     7501 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     7561 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     7621 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     7681 cctggtgcat gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg
     7741 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     7801 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     7861 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     7921 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     7981 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     8041 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     8101 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     8161 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     8221 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     8281 agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta
     8341 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     8401 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     8461 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     8521 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     8581 atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg
     8641 tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa
     8701 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     8761 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     8821 tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc
     8881 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     8941 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg
     9001 acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     9061 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     9121 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     9181 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     9241 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     9301 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     9361 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     9421 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     9481 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     9541 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     9601 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     9661 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     9721 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     9781 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     9841 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     9901 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     9961 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
    10021 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
    10081 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
    10141 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
    10201 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
    10261 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
    10321 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
    10381 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
    10441 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
    10501 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
    10561 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
    10621 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
    10681 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
    10741 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
    10801 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
    10861 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
    10921 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
    10981 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
    11041 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
    11101 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
    11161 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
    11221 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
    11281 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
    11341 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
    11401 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
    11461 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
    11521 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
    11581 agcggaagag
//
